Methods and compositions for killing spores

ABSTRACT

The invention provides a sporocidal composition comprising a laccase or a compound exhibiting laccase activity, a source of oxygen, a source of iodide ions and an enhancing agenL A method of killing or inactivating spores and a method of decontaminating a location, which has been exposed to spores, are also disclosed.

FIELD OF THE INVENTION

The present invention relates to enzymatic methods for killing orinactivating microbial spores.

BACKGROUND

Spores are known to form from aerobic Bacilli, anaerobic Clostridia,selected sarcinae and a few actinomycetes. Spores resemble certain plantseeds in that they do not carry out any metabolic reactions. In thisregard they are especially suited to withstand severe environmentalstress and are known to survive prolonged exposures to heat, drying,radiation and toxic chemicals. These properties make spores especiallydifficult to kill in environments, like living tissue or objects whichcome in contact with living tissue, which would be adversely effected byextreme conditions.

Fungi, viruses and vegetative cells of pathogenic bacteria aresterilized within minutes at 70 degrees Celsius; many spores aresterilized at 100 degrees Celsius. However, the spores of somesaprophytes can survive boiling for hours. Heat is presently the mostcommonly used means to insure sterilization of spores.

A particularly difficult problem relates to microbiocidal treatment ofbacterial spore-forming microorganisms of the Bacillus cereus group.

Microorganisms of the Bacillus cereus group include Bacillus cereus,Bacillus mycoides, Bacillus anthracis, and Bacillus thuringiensis. Thesemicroorganisms share many phenotypical properties, have a high level ofchromosomal sequence similarity, and are known enterotoxin producers.

Although all spore-forming microorganisms are problematic formicrobiocidal treatments because they form spores, Bacillus cereus isone of the most problematic because Bacillus cereus has been identifiedas possessing increased resistance to germicidal chemicals used todecontaminate environmental surfaces.

Bacillus cereus is a particularly well-established enterotoxin producerand food-borne pathogen. This organism is frequently diagnosed as acause of gastrointestinal disorders and has been suggested to be thecause of several foodborne illness outbreaks. The organism is ubiquitousin nature, and as a consequence, is present in animal feed and fodder.Due to its rapid sporulating capacity, the organism easily survives inthe environment and can survive intestinal passage in cows. The organismcan contaminate raw milk via feces and soil, and Bacillus cereus caneasily survive the pasteurization process.

The present invention provides an improved enzymatic method for killingor inactivating spores.

SUMMARY

The present invention provides as a first aspect a sporocidalcomposition comprising a laccase or a compound exhibiting laccaseactivity, a source of oxygen, a source of iodide ions and an enhancingagent.

In a second aspect is provided a method of killing or inactivatingspores, comprising contacting the spores with the sporocidal compositionof the invention.

In a third aspect is provided a method of decontaminating a location,which has been exposed to spores, comprising contacting the spores withthe composition of the invention.

In a fourth aspect is provided a container comprising the composition ofthe invention, wherein the components of the composition are packaged inone or more compartments or layers.

In a fifth aspect is provided a ready-to-use sporocidal formulationcomprising the composition of the invention.

In embodiments, the source of iodide may be one or more salts of iodide,such as sodium iodide or potassium iodide or mixtures thereof.

In other embodiments, the sporocidal composition of the inventionfurther comprises a surfactant.

DETAILED DESCRIPTION

Laccases and Compounds Exhibiting Laccase Activity

Compounds exhibiting laccase activity may be any laccase enzymecomprised by the enzyme classification EC 1.10.3.2 as set out by theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB), or any fragment derived therefrom exhibitinglaccase activity, or a compound exhibiting a similar activity, such as acatechol oxidase (EC 1.10.3.1), an o-aminophenol oxidase (EC 1.10.3.4),or a bilirubin oxidase (EC 1.3.3.5).

Preferred laccase enzymes and/or compounds exhibiting laccase activityare enzymes of microbial origin. The enzymes may be derived from plants,bacteria or fungi (including filamentous fungi and yeasts).

Suitable examples from fungi include a laccase derivable from a strainof Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis,Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizoctonia, e.g., R. solani, Coprinus, e.g., C. cinereus,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g., C.hirsutus (JP 2-238885).

Suitable examples from bacteria include a laccase derivable from astrain of Bacillus.

A laccase derived from Coprinus, Myceliophthora, Polyporus, Scytalidiumor Rhizoctonia is preferred; in particular a laccase derived fromCoprinus cinereus, Myceliophthora thernophila, Polyporus pinsitus,Scytalidium thermophilum or Rhizoctonia solani.

The laccase or the laccase related enzyme may furthermore be one whichis producible by a method comprising cultivating a host cell transformedwith a recombinant DNA vector which carries a DNA sequence encoding saidlaccase as well as DNA sequences encoding functions permitting theexpression of the DNA sequence encoding the laccase, in a culture mediumunder conditions permitting the expression of the laccase enzyme, andrecovering the laccase from the culture.

Determination of Laccase Activity (LACU)

Laccase activity (particularly suitable for Polyporus laccases) may bedetermined from the oxidation of syringaldazin under aerobic conditions.The violet colour produced is photometered at 530 nm. The analyticalconditions are 19 mM syringaldazin, 23 mM acetate buffer, pH 5.5, 30°C., 1 min. reaction time.

1 laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 mmole syringaldazin per minute at these conditions.

Determination of Laccase Activity (LAMU)

Laccase activity may be determined from the oxidation of syringaldazinunder aerobic conditions. The violet colour produced is measured at 530nm. The analytical conditions are 19 mM syringaldazin, 23 mMTristmaleate buffer, pH 7.5, 30° C., 1 min. reaction time.

1 laccase unit (LAMU) is the amount of enzyme that catalyses theconversion of 1.0 mmole syringaldazin per minute at these conditions.

Source of Oxygen

The source of oxygen required by the laccase or the compound exhibitinglaccase activity may be oxygen from the atmosphere or an oxygenprecursor for in situ production of oxygen. Oxygen from the atmospherewill usually be present in sufficient quantity. If more O₂ is needed,additional oxygen may be added, e.g. as pressurized atmospheric air oras pure pressurized O₂.

Source of Iodide Ions

According to the invention the source of iodide ions needed for thereaction with the laccase may be achieved in many different ways, suchas by adding one or more salts of iodide. In a preferred embodiment thesalt of iodide is sodium iodide or potassium iodide, or mixturesthereof.

The concentration of the source of iodide ions will typically correspondto a concentration of iodide ions of from 0.01 mM to 1000 mM, preferablyfrom 0.05 mM to 500 mM, and more preferably from 0.1 mM to 100 mM.

Enhancing Agent

The enhancing agent may be selected from the group consisting ofaliphatic, cyclo-aliphatic, heterocyclic or aromatic compoundscontaining the moiety >N—OH. In a preferred embodiment of the inventionthe enhancing agent is a compound of the general formula I:

wherein R¹, R², R³, R⁴ are individually selected from the groupconsisting of hydrogen, halogen, hydroxy, formyl, carboxy and salts andesters thereof, amino, nitro, C₁₋₁₂-alkyl, C₁₋₆-alkoxy,carbonyl(C₁₋₁₂-alkyl), aryl, in particular phenyl, sulfo, aminosulfonyl,carbamoyl, phosphono, phosphonooxy, and salts and esters thereof,wherein the R¹, R², R³, R⁴ may be substituted with R⁵, wherein R⁵represents hydrogen, halogen, hydroxy, formyl, carboxy and salts andesters thereof, amino, nitro, C₁₋₁₂-alkyl, C₁₋₆-alkoxy,carbonyl(C₁₋₁₂-alkyl), aryl, in particular phenyl, sulfo, aminosuifonyl,carbamoyl, phosphono, phosphonooxy, and salts and esters thereof;

-   -   [X] represents a group selected from (—N═N—)_(m), (—N═CR⁶—)_(m),        (—CR⁶═N—)_(m), (—CR⁷═CR⁶—)_(m)(—CR⁶═N—NR⁷—), (—N═N—CHR⁶—),        (—N═CR⁶—NR⁷—), (—N═CR⁶—CHR⁷—), (—CR⁶═N—CHR⁷—), (—CR⁶═CR⁷—NR⁸—),        and (—CR⁶═CR⁷—CHR⁸ 13 ), wherein R⁶, R⁷, and R⁸ independently of        each other are selected from H, OH, NH₂, COOH, SO₃H, C₁₋₆-alkyl,        NO₂, CN, Cl, Br, F, CH₂OCH₃, OCH₃, and COOCH₃; and m is 1 or 2.

The term “C_(1-n)-alkyl” wherein n can be from 2 through 12, as usedherein, represent a branched or straight alkyl group having from one tothe specified number of carbon atoms. Typical C₁₋₆-alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, hexyl, iso-hexyland the like.

In a more preferred embodiment of the invention the enhancing agent is acompound of the general formula II:

wherein R¹, R², R³, R⁴ are individually selected from the groupconsisting of hydrogen, halogen, hydroxy, formyl, carboxy and salts andesters thereof, amino, nitro, C₁₋₁₂-alkyl, C₁₋₆-alkoxy,carbonyl(C₁₋₁₂-alkyl), aryl, in particular phenyl, sulfo, aminosulfonyl,carbamoyl, phosphono, phosphonooxy, and salts and esters thereof,wherein the R¹, R², R³, R⁴ may be substituted with R⁵, wherein R⁵represents hydrogen, halogen, hydroxy, formyl, carboxy and salts andesters thereof, amino, nitro, C₁₋₁₂-alkyl, C₁₋₆-alkoxy,carbonyl(C₁₋₁₂-alkyl), aryl, in particular phenyl, sulfo, aminosulfonyl,carbamoyl, phosphono, phosphonooxy, and salts and esters thereof.

The enhancing agent may also be a salt or an ester of formula I or II.

Further preferred enhancing agents are oxoderivatives and N-hydroxyderivatives of heterocyclic compounds and oximes of oxo- andformyl-derivatives of heterocyclic compounds, said heterocycliccompounds including five-membered nitrogen-containing heterocycles, inparticular pyrrol, pyrazole and imidazole and their hydrogenatedcounterparts (e.g. pyrrolidine) as well as triazoles, such as1,2,4-triazole; six-membered nitrogen-containing heterocycles, inparticular mono-, di- and triazinanes (such as piperidine andpiperazine), morpholine and their unsaturated counterparts (e.g.pyridine and pyrimidine); and condensed heterocycles containing theabove heterocycles as substructures, e.g. indole, benzothiazole,quinoline and benzoazepine.

Examples of preferred enhancing agent from these classes of compoundsare pyridine aldoximes; N-hydroxypyrrolidinediones such , asN-hydroxysuccinimide and N-hydroxyphthalimide;3,4-dihydro-3-hydroxybenzo[1,2,3]triazine-4-one; formaldoxime trimer(N,N′,N″-trihydroxy-1,3,5-triazinane); and violuricacid(1,3-diazinane-2,4,5,6-tetrone-5-oxime).

Still further enhancing agents which may be applied in the inventioninclude oximes of oxo- and formyl-derivatives of aromatic compounds,such as benzoquinone dioxime and salicylaldoxime (2-hydroxybenzaldehydeoxime), and N-hydroxyamides and N-hydroxyanilides, such asN-hydroxyacetanilide.

Preferred enhancing agents are selected from the group consisting of1-hydroxybenzotriazole; 1-hydroxybenzotriazole hydrate;1-hydroxybenzotriazole sodium salt; 1-hydroxybenzotriazole potassiumsalt; 1-hydroxybenzotriazole lithium salt; 1-hydroxybenzotriazoleammonium salt; 1-hydroxybenzotriazole calcium salt;1-hydroxybenzotriazole magnesium salt; and1-hydroxybenzotriazole-6-sulphonic acid.

A particularly preferred enhancing agent is 1-hydroxybenzotriazole.

All the specifications of N-hydroxy compounds above are understood toinclude tautomeric forms such as N-oxides whenever relevant.

Another preferred group of enhancing agents comprises a —CO—NOH— groupand has the general formula III:

in which A is:

and B is the same as A; or B is H or C₁₋₁₂-alkyl, said alkyl may containhydroxy, ester or ether groups (e.g. wherein the ether oxygen isdirectly attached to A-N(OH)C═O—, thus including N—hydroxy carbamic acidester derivatives), and R2, R3, R4, R5 and R6 independently of eachother are H, OH, NH₂, COOH, SO₃H, C₁₋₈-alkyl, acyl, NO₂, CN, Cl, Br, F,CF₃, NOH—CO-phenyl, CO—NOH-phenyl, C₁₋₆—CO—NOH-A, CO—NOH-A, COR12,phenyl-CO—NOH-A, OR7, NR8R9, COOR10, or NOH—CO—R11, wherein R7, R8, R9,R10, R 11 and R12 are C₁₋₁₂-alkyl or acyl.

R2, R3, R4, R5 and R6 of A are preferably H, OH, NH₂, COOH, SO₃H,C₁₋₃-alkyl, acyl, NO₂, CN, Cl, Br, F, CF₃, NOH—CO-phenyl, CO—NOH-phenyl,COR12, OR7, NR8R9, COOR10, or NOH—CO—R11, wherein R7, R8 and R9 areC₁₋₃-alkyl or acyl, and R10, R11 and R12 are C₁₋₃-alkyl; more preferablyR2, R3, R4, R5 and R6 of A are H, OH, NH₂, COOH, SO₃H, CH₃, acyl, NO₂,CN, Cl, Br, F, CF₃, CO—NOH-phenyl, COCH₃, OR7, NR8R9, or COOCH₃, whereinR7, R8 and R9 are CH₃ or COCH₃; even more preferably R2, R3, R4, R5 andR6 of A are H, OH, COOH, SO₃H, CH₃, acyl, NO₂, CN, Cl, Br, F,CO—NOH-phenyl, OCH₃, COCH₃, or COOCH₃; and in particular R2, R3, R4, R5and R6 of A are H, OH, COOH, SO₃H, CH₃, NO₂, CN, Cl, Br, CO—NOH-phenyl,or OCH₃.

R2, R3, R4, R5 and R6 of B are preferably H, OH, NH₂, COOH, SO₃H,C₁₋₃-alkyl, acyl, NO₂, CN, Cl, Br, F, CF₃, NOH—CO-phenyl, CO—NOH-phenyl,COR12, OR7, NR8R9, COOR10, or NOH—CO—R11, wherein R7, R8 and R9 areC₁₋₃-alkyl or acyl, and R10, R11 and R12 are C₁₋₃-alkyl; more preferablyR2, R3, R4, R5 and R6 of B are H, OH, NH₂, COOH, SO₃H, CH₃, NO₂, CN, Cl,Br, F, CF₃, CO—NOH-phenyl, COCH₃, OR7, NR8R9, or COOCH₃, wherein R7, R8and R9 are CH₃ or COCH₃; even more preferably R2, R3, R4, R5 and R6 of Bare H, OH, COOH, SO₃H, CH₃, acyl, NO₂, CN, Cl, Br, F, CO—NOH-phenyl,OCH₃, COCH₃, or COOCH₃; and in particular R2, R3, R4, R5 and R6 of B areH, OH, COOH, SO₃H, CH₃, NO₂, CN, Cl, Br, CO—NOH-phenyl, or OCH₃.

B is preferably H or C₁₋₃-alkyl, said alkyl may contain hydroxy, esteror ether groups; preferably said alkyl may contain ester or ethergroups; more preferably said alkyl may contain ether groups.

In an embodiment, A and B independently of each other are:

or B is H or C₁₋₃-alkyl, said alkyl may contain hydroxy, ester or ethergroups (e.g. wherein the ether oxygen is directly attached toA-N(OH)C═O—, thus including N-hydroxy carbamic acid ester derivatives),and R2, R3, R4, R5 and R6 independently of each other are H, OH, NH₂,COOH, SO₃H, C₁₋₃-alkyl, acyl, NO₂, CN, Cl, Br, F, CF₃, NOH—CO-phenyl,CO—NOH-phenyl, COR12, OR7, NR8R9, COOR10, or NOH—CO—R11, wherein R7, R8and R9 are C₁₋₃-alkyl or acyl, and R10, R11 and R12 are C₁₋₃-alkyl.

In another embodiment, A and B independently of each other are:

or B is H or C₁₋₃-alkyl, said alkyl may contain hydroxy or ether groups(e.g. wherein the ether oxygen is directly attached to A-N(OH)C═O—, thusincluding N-hydroxy carbamic acid ester derivatives), and R2, R3, R4, R5and R6 independently of each other are H, OH, NH₂, COOH, SO₃H, CH₃,acyl, NO₂, CN, Cl, Br, F, CF₃, CO—NOH-phenyl, COCH₃, OR7, NR8R9, orCOOCH₃, wherein R7, R8 and R9 are CH₃ or COCH₃.

In another embodiment, A and B independently of each other are:

or B is H or C₁₋₃-alkyl, said alkyl may contain hydroxy or ether groups(e.g. wherein the ether oxygen is directly attached to A-N(OH)C═O—, thusincluding N-hydroxy carbamic acid ester derivatives), and R2, R3, R4, R5and R6 independently of each other are H, OH, COOH, SO₃H, CH₃, acyl,NO₂, CN, Cl, Br, F, CO—NOH-phenyl, OCH₃, COCH₃, or COOCH₃.

In another embodiment, A and B independently of each other are:

or B is C₁₋₃-alkyl, said alkyl may contain ether groups (e.g. whereinthe ether oxygen is directly attached to A-N(OH)C═O—, thus includingN-hydroxy carbamic acid ester derivatives), and R2, R3, R4, R5 and R6independently of each other are H, OH, COOH, SO₃H, CH₃, NO₂, CN, Cl, Br,CO—NOH-phenyl, or OCH₃.

The terms “C_(1-n)-alkyl” wherein n can be from 2 through 12, as usedherein, represent a branched or straight alkyl group having from one tothe specified number of carbon atoms. Typical C₁₋₆-alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, hexyl, iso-hexyland the like.

The term “acyl” as used herein refers to a monovalent substituentcomprising a C₁₋₆-alkyl group linked through a carbonyl group; such ase.g. acetyl, propionyl, butyryl, isobutyryl, pivaloyl, valeryl, and thelike.

In an embodiment at least one of the substituents R2, R3, R4, R5 and R6of A are H, preferably at least two of the substituents R2, R3, R4, R5and R6 of A are H, more preferably at least three of the substituentsR2, R3, R4, R5 and R6 of A are H, most preferably at least four of thesubstituents R2, R3, R4, R5 and R6 of A are H, in particular all of R2,R3, R4, R5 and R6 of A are H.

In another embodiment at least one of the substituents R2, R3, R4, R5and R6 of B are H, preferably at least two of the substituents R2, R3,R4, R5 and R6 of B are H, more preferably at least three of thesubstituents R2, R3, R4, R5 and R6 of B are H, most preferably at leastfour of the substituents R2, R3, R4, R5 and R6 of B are H, in particularall of R2, R3, R4, R5 and R6 of B are H.

In particular embodiments according to the invention the enhancing agentis selected from the group consisting of

-   -   4-nitrobenzoic acid-N-hydroxyanilide;    -   4-methoxybenzoic acid-N-hydroxyanilide;    -   N,N′-dihydroxy-N,N′-diphenylterephthalamide;    -   decanoic acid-N-hydroxyanilide;    -   N-hydroxy-4-cyanoacetanilide;    -   N-hydroxy-4-acetylacetanilide;    -   N-hydroxy-4-hydroxyacetanilide;    -   N-hydroxy-3-(N′-hydroxyacetamide)acetanilide;    -   4-cyanobenzoic acid-N-hydroxyanilide;    -   N-hydroxy-4-nitroacetanilide;    -   N-hydroxyacetanilide;    -   N-hydroxy-N-phenyl-carbamic acid isopropyl ester;    -   N-hydroxy-N-phenyl-carbamic acid methyl ester;    -   N-hydroxy-N-phenyl-carbamic acid phenyl ester;    -   N-hydroxy-N-phenyl-carbamic acid ethyl ester; and    -   N-hydroxy-N-(4-cyanophenyl)-carbamic acid methyl ester.

Another group of preferred enhancing agents is phenolic compounds(alkylsyringates) of the general formula IV:

wherein the letter A in said formula denotes be a group such as -D,—CH═CH-D, —CH═CH—CH═CH-D, —CH═N-D, —N═N-D, or —N═CH-D, in which D isselected from the group consisting of —CO-E, —SO₂-E, —N—XY, and —N⁺—XYZ,in which E may be —H, —OH, —R, or —OR, and X and Y and Z may beidentical or different and selected from —H and —R; R being a C₁-C₁₆alkyl, preferably a C₁-C₈ alkyl. which alkyl may be saturated orunsaturated, branched or unbranched and optionally substituted with acarboxy, sulpho or amino group; and B and C may be the same or differentand selected from C_(m)H_(2m+1), where m=1, 2, 3, 4 or 5.

In the above mentioned general formula IV, A may be placed meta to thehydroxy group instead of being placed in the para-position as shown.

In particular embodiments of the invention the enhancing agent isselected from the group having the general formula V:

in which A is a group such as —H, —OH, —CH₃, —OCH₃, —O(CH₂)_(n)CH₃,where n=1, 2, 3, 4, 5, 6, 7 or 8.

Yet another group of preferred enhancing agents are the compounds asdescribed in general formula VI:

in which general formula A represents a single bond, or one of thefollowing groups: (—CH₂—), (—CH═CH—), (—NR11-), (—CH═N—), (—N═N—),(—CH═N—N═CH—), or (>C═O);

-   -   and in which general formula the substituent groups R1-R11,        which may be identical or different, independently represents        any of the following radicals: hydrogen, halogen, hydroxy,        formyl, acetyl, carboxy and esters and salts hereof, carbamoyl,        sulfo and esters and salts hereof, sulfamoyl, methoxy, nitro,        amino, phenyl, C₁₋₈-alkyl;    -   which carbamoyl, sulfamoyl, phenyl, and amino groups may        furthermore be unsubstituted or substituted once or twice with a        substituent group R12; and which C₁₋₈-alkyl group may be        saturated or unsaturated, branched or unbranched, and may        furthermore be unsubstituted or substituted with one or more        substituent groups R12;    -   which substituent group R12 represents any of the following        radicals: hydrogen, halogen, hydroxy, formyl, acetyl, carboxy        and esters and salts hereof, carbamoyl, sulfo and esters and        salts hereof, sulfamoyl, methoxy, nitro, amino, phenyl, or        C₁₋₈-alkyl; which carbamoyl, sulfamoyl, and amino groups may        furthermore be unsubstituted or substituted once or twice with        hydroxy or methyl.    -   and in which general formula R5 and R6 may together form a group        —B—, in which B represents a single bond, one of the following        groups (—CH₂—), (—CH═CH—), (—CH═N—); or B represents sulfur, or        oxygen.

In particular embodiments of the invention the enhancing agent isselected from the group having the general formula VIl:

in which general formula X represents a single bond, oxygen, or sulphur;

-   -   and in which general formula the substituent groups R1-R9, which        may be identical or different, independently represents any of        the following radicals: hydrogen, halogen, hydroxy, formyl,        acetyl, carboxy and esters and salts hereof, carbamoyl, sulfo        and esters and salts hereof, sulfamoyl, methoxy, nitro, amino,        phenyl, C₁₋₈-alkyl;    -   which carbamoyl, sulfamoyl, phenyl, and amino groups may        furthermore be unsubstituted or substituted once or twice with a        substituent group R10; and which C₁₋₈-alkyl group may be        saturated or unsaturated, branched or unbranched, and may        furthermore be unsubstituted or substituted with one or more        substituent groups R10;    -   which substituent group R10 represents any of the following        radicals: hydrogen, halogen, hydroxy, formyl, acetyl, carboxy        and esters and salts hereof, carbamoyl, sulfo and esters and        salts hereof, sulfamoyl, methoxy, nitro, amino, phenyl, or        C₁₋₈-alkyl; which carbamoyl, sulfamoyl, and amino groups may        furthermore be unsubstituted or substituted once or twice with        hydroxy or methyl.

According to the invention, the enhancing agent may be present in thecomposition in a concentration in the range of from 0.01 mM to 1000 mM,preferably in the range of from 0.05 mM to 500 mM, more preferably inthe range of from 0.1 mM to 100 mM, and most preferably in the range offrom 0.1 mM to 50 mM.

Spores

The spores which are contacted with a laccase or a compound exhibitinglaccase activity, a source of oxygen, a source of iodide ions and anenhancing agent in the method of the invention comprise all kinds ofspores.

In an embodiment the spores are endospores, such as all Clostridium sp.spores, Brevibacillus sp. spores and Bacillus sp. spores, e.g. sporesfrom Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillusthuringiensis, Bacillus subtilis, Bacillus putida, and Bacillus pumila.

In another embodiment the spores are exospores, such as Actinomycetalesspores, e.g. spores from Actinomyces sp., Streptomyces sp.,Thermoactinomyces sp., Saccharomonospora sp., and Saccharopylospora sp.

In another embodiment the spores are bacterial spores. Examples ofbacterial spores include, but are not limited to, all Clostridium sp.spores and Bacillus sp. spores as mentioned above.

In yet another embodiment the spores are fungal spores. Examples offungal spores include (in addition to those mentioned above), but arenot limited to, conidiospores, such as spores from Aspergillus sp., andPenicillium sp.

Surfactants

The surfactants suitable for being incorporated in the sporocidalcomposition may be non-ionic (including semi-polar), anionic, cationicand/or zwitterionic. The surfactants are preferably anionic ornon-ionic. The surfactants are typically present in the sporocidalcomposition at a concentration of from 0.01% to 10% by weight.

When included therein, the sporocidal composition will usually containfrom about 0.01% to about 10%, preferably about 0.05% to about 5%, andmore preferably about 0.1% to about 1% by weight of an anionicsurfactant, such as linear alkylbenzenesulfonate, alpha-olefinsulfonate,alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondaryalkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- oralkenylsuccinic acid or soap.

When included therein the sporocidal composition will usually containfrom about 0.01% to about 10%, preferably about 0.05% to about 5%, andmore preferably about 0.1% to about 1% by weight of a non-ionicsurfactant, such as alcohol ethoxylate, nonylphenol ethoxylate,alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acidmonoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fattyacid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).

Compositions

The present invention provides a composition comprising a laccase or acompound exhibiting laccase activity, a source of oxygen, a source ofiodide ions and an enhancing agent.

The laccase or the compound exhibiting laccase activity, the source ofiodide ions and the enhancing agent may be formulated as a liquid (e.g.aqueous), a solid, a gel, a paste or a dry product formulation. The dryproduct formulation may subsequently be re-hydrated to form an activeliquid or semi-liquid formulation usable in the method of the invention.

When the laccase or the compound exhibiting laccase activity, the sourceof iodide ions and the enhancing agent are formulated as a dryformulation, the components may be mixed, arranged in discrete layers orpackaged separately.

When formulated as a solid, all components may be mixed together, e.g.,as a powder, a granulate or a gelled product.

When other than dry form compositions are used and even in that case, itis preferred to use a two-part formulation system having the enzyme(s)separate from the rest of the composition.

The composition of the invention may further comprise auxiliary agentssuch as wetting agents, thickening agents, buffer, stabilisers, perfume,colourants, fillers and the like.

Useful wetting agents are surfactants, i.e. non-ionic, anionic,amphoteric or zwitterionic surfactants. Surfactants are furtherdescribed above.

The composition of the invention may be a concentrated product or aready-to-use product. In use, the concentrated product is typicallydiluted with water to provide a medium having an effective sporocidalactivity, applied to the object to be cleaned or disinfected, andallowed to react with the spores present.

The pH of an aqueous solution of the composition is in the range of frompH 2 to 11, preferably in the range of from pH 3 to 10.5, morepreferably in the range of from pH 4 to 10, most preferably in the rangeof from pH 5 to 9, and in particular in the range of from pH 6 to 8.

Methods and Uses

The present invention provides an enzymatic method for killing orinactivating spores, comprising contacting the spores with a laccase ora compound exhibiting laccase activity, a source of oxygen, a source ofiodide ions and an enhancing agent.

In the context of the present invention the term “killing orinactivating spores” is intended to mean that at least 99% of the sporesare not capable of transforming (germinating) into vegetative cells.Preferably 99.9% (more preferably 99.99% and most preferably 99.999%) ofthe spores are not capable of transforming into vegetative cells.

The spores May be contacted by the composition of the invention at atemperature between 0 and 90 degrees Celsius, preferably between 5 and80 degrees Celsius, more preferably between 10 and 70 degrees Celsius,even more preferably between 15 and 60 degrees Celsius, most preferablybetween 18 and 50 degrees Celsius, and in particular between 20 and 40degrees Celsius.

The composition of the invention is suitable for killing or inactivatingspores in a variety of environments. The composition of the inventionmay desirably be used in any environment to reduce spore contamination,such as the health-care industry (e.g. animal hospitals, humanhospitals, animal clinics, human clinics, nursing homes, day-carefacilities for children or senior citizens, etc.), the food industry(e.g. restaurants, food-processing plants, food-storage plants, grocerystores, etc.), the hospitality industry (e.g. hotels, motels, resorts,cruise ships, etc.), the education industry (e.g. schools anduniversities), etc.

The composition of the invention may desirably be used in anyenvironment to reduce spore contamination, such as general-premisesurfaces (e.g. floors, walls, ceilings, exterior of furniture, etc.),specific-equipment surfaces (e.g. hard surfaces, manufacturingequipment, processing equipment, etc.), textiles (e.g. cottons, wools,silks, synthetic fabrics such as polyesters, polyolefins, and acrylics,fiber blends such as cottonpolyester, etc.), wood and cellulose-basedsystems (e.g. paper), soil, animal carcasses (e.g. hide, meat, hair,feathers, etc.), foodstuffs (e.g. fruits, vegetables, nuts, meats,etc.), and water.

In one embodiment, the method of the invention is directed to sporocidaltreatment of textiles. Spores of the Bacillus cereus group have beenidentified as the predominant postlaundering contaminant of textiles.Thus, the treatment of textiles with a composition of the invention isparticularly useful for sporocidal activity against the contaminants oftextiles.

Examples of textiles that can be treated with the composition of theinvention include, but are not limited to, personal items (e.g. shirts,pants, stockings, undergarments, etc.), institutional items (e.g.towels, lab coats, gowns, aprons, etc.), hospitality items (e.g. towels,napkins, tablecloths, etc.).

A sporocidal treatment of textiles with a composition of the inventionmay include contacting a textile with a composition of the invention.This contacting can occur prior to laundering the textile.Alternatively, this contacting can occur during laundering of thetextile to provide sporocidal activity and optionally provide cleansingactivity to remove or reduce soils, stains, etc. from the textile.

The spores which are contacted by the composition of the invention maybe situated on any surface including, but not limited to, a surface of aprocess equipment used in e.g. a dairy, a chemical or pharmaceuticalprocess plant, a piece of laboratory equipment, a water sanitationsystem, an oil processing plant, a paper pulp processing plant, a watertreatment plant, or a cooling tower. The composition of the inventionshould be used in an amount, which is effective for killing orinactivating the spores on the surface in question.

The spores may be contacted with the composition of the invention bysubmerging the spores in an aqueous formulation of the composition (e.g.a laundering process), by spraying the composition onto the spores, byapplying the composition to the spores by means of a cloth, or by anyother method recognized by the skilled person. Any method of applyingthe composition of the invention to the spores, which results in killingor inactivating the spores, is an acceptable method of application.

The method of the invention is also useful for decontamination oflocations which have been exposed to spores (e.g. pathogenic spores),such as biological warfare agents, e.g. spores of Bacillus anthrasiscapable of causing anthrax. Such locations include, but are not limitedto, clothings (such as army clothings), inner and outer parts ofvehicles, inner and outer parts of buildings, any kind of army facility,and any kind of environment mentioned above.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Example 1

Production of Spores

A Tryptose Blod Agar Base (TBAB) plate was streaked from a fresh cultureof Bacillus thuringiensis (B. thuringiensis type strain ATCC10792). Theculture was incubated overnight at 30 degrees Celsius.

A loopfull of pure B. thuringiensis cells from the TBAB plate wassuspended in 2 ml of sterile water. 2×SG plates were each inoculatedwith 100 microliter of the cell suspension. The composition of 2×SG wasas follows: 16 g/L Difco Bacto Nutrient Broth, 0.5 MgSO₄×7H₂O, 2.0 g/LKCl, 1.0 ml/100 ml of 10% glucose, 0.1 ml/100 ml of 1 M Ca(NO₃)₂, 0.1ml/100 ml of 0.1 M MnSO₄, 10 microliter/100 ml of 0.01 M FeSO₄, and 1%Difco Bacto Agar.

Plates were incubated for 48-72 hrs. at 30 degrees Celsius. Sporulationwas checked with phase-contrast microscopy. Spores are phase-bright.

When sporulation efficiency was close to 100%, the cell lawn washarvested with water and the cells were suspended by intensivevortexing. Cells were collected by-centrifugation for 5-10 minutes at6000 G at 4 degrees Celsius, and washed 3 times with ice cold water. Thepellet contained vegetative cells and spores.

A step-density gradient was applied for separation of the spores fromthe vegetative cells. A centrifuge tube containing 30 ml 43% Urographin®was prepared for each washed pellet. 3 ml of cell spore mixture inUrographin was prepared so that the final Urographin concentration was20%. The 20% Urographin cell/spore mixture was gently loaded onto thetop layer of the centrifuge tubes containing 43% Urographin.

The centrifuge tubes were centrifuged at 10000 G at room temperature for30 minutes. The supernatant was gently removed. The pure spore pelletwas suspended in 1 ml ice-cold water and transfered to a microfuge tube.Centrifugation was continued at maximum speed for 1-2 min at 4 degreesCelsius, and the pellet was washed in ice-cold water 2 more times.

The purity and number of spores/ml was checked by phase contrastmicroscopy and a haemocytometer. The spores were stored suspended inwater at minus 20 degrees Celsius.

Bacillus globigii spores were produced by following the same procedureas outlined above.

Example 2

Killing of Spores

The following reagents were prepared:

-   -   DMG buffer (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.5 with NaOH;    -   Bacillus thuringiensis spores were re-suspended in DMG buffer to        a density of 2×10⁹ spores per ml;    -   Myceliophthora thermophila laccase (as disclosed in WO 95/33836,        SEQ ID NO:1; and available from Novozymes ANS) was diluted to        200 microgram per ml in DMG buffer;    -   Polyporus pinsitus laccase (as disclosed in WO 96/00290, FIG. 1,        SEQ ID NO:1; and available from Novozymes ANS) was diluted to        200 microgram per ml in DMG buffer;    -   200 mM Potassium iodide (KI) solution in water;    -   1 mM Methylsyringate (methyl 3,5-dimethoxy4-hydroxybenzoate,        Sigma S40,944-8) solution in ethanol/DMG buffer (1:1);    -   3 mM MTT (3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium        bromide, Sigma M2128) solution in water.

TBB growth medium:

-   -   10 g/l Tryptose,    -   3 g/l Beef Extract,    -   5 g/l NaCl,    -   water ad 1000 ml    -   final pH 7.2+/−0.2.

Spore suspension was pipetted into the wells in row A of a microtiterplate. The other reagents were added as indicated in table 1 below. Thereaction was initiated by the addition of laccase solution. TABLE 1 DMGbuffer Spores Laccase KI Methyl- (micro- (micro- (micro- (micro-syringate Wells liter) liter) liter) liter) (microliter) A1-A2 115 50 205 10 A3-A4 120 50 20 0 10 A5-A6 125 50 20 5 0 A7-A8 145 50 0 5 0  A9-A10130 50 20 0 0 A11-A12 150 50 0 0 0

The microtiter plate was incubated at room temperature (24 degreesCelsius) for 1 hour. 180 microliter TBB growth medium was added to allwells in rows B to H of the microtiter plate. Serial 10 fold dilutionswere made by pipetting 20 microliter from row A to row B, and then fromrow B to row C, and then from row C to row D, and so on until row H.

The microtiter plate was incubated at 30 degrees Celsius for 20-24 hoursto allow spores to germinate and grow. Growth was evaluated by amicroplate reader and visually by “developing the growth” by addition of5 microliter 3 mM MTT to each well. Formation of purple formazansreveals bacterial growth and thus the degree of spore inactivation. Inthe tables growth is indicated with a “+” symbol. TABLE 2 Results ofevaluation of growth for spores treated with Myceliophthora thermophilalaccase. 1 2 3 4 5 6 7 8 9 10 11 12 A B − − + + + + + + + + − + C −− + + + + + + + + + + D − − + + + + + + + + − + E − − + + + + + + + +− + F − − + + + + + + + + − + G − − + + + + + + + + − + H − − − − − − −− + + − −

TABLE 3 Results of evaluation of growth for spores treated withPolyporus pinsitus laccase. 1 2 3 4 5 6 7 8 9 10 11 12 A B −− + + + + + + + + + + C − − + + + + + + + + + + D −− + + + + + + + + + + E − − + + + + + + + + + + F −− + + + + + + + + + + G − − + − + + + + + + + + H − − − − − − + − + + −+

The results in Tables 2 and 3 show that only the formulation added towells A1-A2 including both laccase, potassium iodide and enhancing agent(methylsyringate) is capable of inactivating the spores.

Example 3

Killing of Spores at 5-60 Degrees Celsius

The following reagents were prepared:

-   -   DMG buffer (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.5 with NaOH;    -   Bacillus thuringiensis spores were re-suspended in DMG buffer to        a density of 2×10⁹ spores per ml;    -   Myceliophthora thermnophila laccase (as disclosed in WO        95/33836, SEQ ID NO:1; and available from Novozymes A/S) was        diluted to 500 microgram per ml in DMG buffer;    -   Coprinus cinereus laccase (as disclosed in WO 97/08325, FIG. 1,        SEQ ID NO:27; and available from Novozymes A/S) was diluted to        500 microgram per ml in DMG buffer;    -   Rhizoctonia solanii laccase (as disclosed in WO 95/07988, FIG.        4, SEQ ID NO:14; and available from Novozymes A/S) was diluted        to 500 microgram per ml in DMG buffer;    -   Polyporus pinsitus laccase (as disclosed in WO 96/00290, FIG. 1,        SEQ ID NO:1; and available from Novozymes A/S) was diluted to        500 microgram per ml in DMG buffer;    -   200 mM Potassium iodide (KI) solution in water;    -   1 mM Methylsyringate (methyl 3,5-dimethoxy-4-hydroxybenzoate,        Sigma S40,944-8) solution in ethanol/DMG buffer (1:1);    -   3 mM MTT (3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium        bromide, Sigma M2128) solution in water.

TBB growth medium:

-   -   10 g/l Tryptose,    -   3 g/l Beef Extract,    -   5 g/l NaCl,    -   water ad 1000 ml    -   final pH 7.2±0.2.

Spore suspension was pipetted into the wells in row A of all microtiterplates. The other reagents were added as indicated in table 4 below. Thereaction was initiated by the addition of laccase solution. Themicrotiter plates were then incubated at the specified temperature for 1hour.

180 microliter TBB growth medium was added to all wells in rows B to Hof the microtiter plates.

Serial 10 fold dilutions were made by pipetting 20 microliter from row Ato row B, and then from row B to row C, and then from row C to row D,and so on until row H. TABLE 4 Microtiter plate setup - each plate wasused to test two laccases at one temperature. DMG buffer Spores LaccaseKI Methyl- (micro- (micro- (micro- (micro- syringate Wells liter) liter)liter) liter) (microliter) A1-A3 166 50 15 6.25 12.5 A4-A6 200 50 0 0 0A7-A9 166 50 15 6.25 12.5 A10-A12 200 50 0 0 0

The microtiter plates were incubated at 30 degrees Celsius for 20-24hours to allow spores to germinate and grow. Growth was evaluated by amicroplate reader and visually by “developing the growth” by addition of5 microliter 3 mM MTT to each well. Formation of purple formazansreveals bacterial growth and thus the degree of spore inactivation. Thesporocidal potential was calculated as the difference of the number ofdilution steps with bacterial growth between the control and thelaccase/iodide/methylsyringate containing wells. The sporocidalpotential is measured in log units (log U)—one log unit equals adifference in growth of one 10-fold dilution step as described inExample 2.

The results from testing the four laccases at 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55 and 60 degrees Celsius are summarised in tables 5, 6, 7and 8. TABLE 5 Sporocidal effect of Coprinus cinereus laccase at 5-60degrees Celsius. Temperature 5 10 15 20 25 30 35 40 45 50 55 60 (degreesCelsius) Kill, log U 1 1 1 4 4.5 4 4 4.5 4 3.5 3.5 2

TABLE 6 Sporocidal effect of Myceliophthora thermophila laccase at 5-60degrees Celsius. Temperature 5 10 15 20 25 30 35 40 45 50 55 60 (degreesCelsius) Kill, log U 0 3 3 3 5 5.5 5 5 5 3.5 3 3

TABLE 7 Sporocidal effect of Polyporus pinsitus laccase at 5-60 degreesCelsius. Temperature 5 10 15 20 25 30 35 40 45 50 55 60 (degreesCelsius) Kill, log U 3 3.5 6 6.5 7 7 7 7 6 6 5 5

TABLE 8 Sporocidal effect of Rhizoctonia solanii laccase at 5-60 degreesCelsius. Temperature 5 10 15 20 25 30 35 40 45 50 55 60 (degreesCelsius) Kill, log U 1 1.5 3 3 3 3 3 3 2 2 2 2

The results shown in tables 5-8 indicate that all four laccases exhibitsporocidal activity and that the optimal sporocidal effect is deliveredin the temperature range 15-45 degrees Celsius.

Example 4

Killing of Spores at pH 6.0-pH8.0

The following reagents were prepared:

-   -   DMG buffers (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.0, 6.5, 7.0, 7.5 and 8.0 with NaOH;    -   Bacillus thuringiensis spores were re-suspended in DMG buffer to        a density of 2×10⁹ spores per ml;    -   Myceliophthora thermophila laccase (as disclosed in WO 95/33836,        SEQ ID NO:1; and available from Novozymes A/S) was diluted to        500 microgram per ml in DMG buffer;    -   Coptinus cinereus laccase (as disclosed in WO 97/08325, FIG. 1,        SEQ ID NO:27; and available from Novozymes A/S) was diluted to        500 microgram per ml in DMG buffer;    -   Rhizoctonia solanii laccase (as disclosed in WO 95/07988, FIG.        4, SEQ ID NO:14; and available from Novozymes A/S) was diluted        to 500 microgram per ml in DMG buffer;    -   Polyporus pinsitus laccase (as disclosed in WO 96/00290, FIG. 1,        SEQ ID NO:1; and available from Novozymes A/S) was diluted to        500 microgram per ml in DMG buffer;    -   200 mM Potassium iodide (KI) solution in water;    -   1 mM Methylsyringate (methyl 3,5-dimethoxy4-hydroxybenzoate,        Sigma S40,944-8) solution in ethanol/DMG buffer (1:1);    -   3 mM MTT (3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium        bromide, Sigma M2128) solution in water.

TBB growth medium:

-   -   10 g/l Tryptose,    -   3 g/l Beef Extract,    -   5 g/l NaCl,    -   water ad 1000 ml    -   final pH 7.2±0.2.

Spore suspension was pipetted into the wells in row A of a microtiterplate. The other reagents were added as indicated in table 9 below. Thereaction was initiated by the addition of laccase solution. Themicrotiter plate was then incubated at 30 degrees Celsius for 1 hour.

180 microliter TBB growth medium was added to all wells in rows B to Hof the microtiter plate. Serial 10 fold dilutions were made by pipetting20 microliter from row A to row B, and then from row B to row C, andthen from row C to row D, and so on until row H. TABLE 9 Amicrotiterplate was set up for each of the four laccases at each pHvalue. DMG buffer Spores Laccase KI Methyl- (micro- (micro- (micro-(micro- syringate Wells liter) liter) liter) liter) (microliter)  A1-A2182 50 5 6.5 6.5  A3-A4 165.5 50 15 6.5 13  A5-A6 188.5 50 5 6.5 0 A7-A8 188.5 50 5 0 6.5  A9-A10 187 50 0 6.5 6.5 A11-A12 200 50 0 0 0

The microtiter plate was incubated at 30 degrees Celsius for 20-24 hoursto allow spores to germinate and grow. Growth was evaluated by amicroplate reader and visually by “developing the growth” by addition of5 microliter 3 mM MTT to each well. Formation of purple formazansreveals bacterial growth and thus the degree of spore inactivation. Thesporocidal potential was calculated as the difference of the number ofdilution steps (with bacterial growth) between the control and thelaccase/iodide/methylsyringate containing wells. The sporocidalpotential is measured in log units (log U)—one log unit equals adifference in growth of one 10-fold dilution step. The results fromtesting the four laccases at pH 6.0—pH 8.0 are summarised in tables 10,11, 12 and 13. TABLE 10 Sporocidal effect of Coprinus cinereus laccasein the pH range pH 6.0-8.0 PH 6.0 6.5 7.0 7.5 8.0 Kill, log U 5 4.5 3.53.5 2

TABLE 11 Sporocidal effect of Myceliophthora thermophila laccase in thepH range pH 6.0-8.0 pH 6.0 6.5 7.0 7.5 8.0 Kill, log U 6 4 4.5 2.5 2

TABLE 12 Sporocidal effect of Polyporus pinsitus laccase in the pH rangepH 6.0-8.0 pH 6.0 6.5 7.0 7.5 8.0 Kill, log U 7 7 5.5 3 0

TABLE 13 Sporocidal effect of Rhizoctonia solanii laccase in the pHrange pH 6.0-8.0. pH 6.0 6.5 7.0 7.5 8.0 Kill, log U 3.5 3 3 2 0.5

The results in tables 10-13 demonstrate that all 4 laccases are activein the specified pH range.

Example 5

Killing of Spores Deposited on Ceramic Tiles I

The following reagents were prepared:

-   -   DMG buffer (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.5 with NaOH;    -   Bacillus thuringiensis spores were re-suspended in DMG buffer to        a density of 2×10⁹ spores per ml;    -   Myceliophthora thermophila laccase (as disclosed in WO 95/33836,        SEQ ID NO:1; and available from Novozymes A/S) was diluted to 30        mg/ml.    -   200 mM Potassium iodide (KI) solution in water;    -   10 mM Methylsyringate (methyl 3,5-dimethoxy-4-hydroxybenzoate,        Sigma S40,944-8) solution in ethanol/DMG buffer (1:1);    -   3 mM MTT (3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium        bromide, Sigma M2128) solution in water.

TBB growth medium with agarose:

-   -   10 g/l Tryptose,    -   3 g/l Beef Extract,    -   5 g/l NaCl,    -   5 g/l Agarose    -   water ad 1000 ml    -   final pH 7.2+/−0.2.

Spores were diluted to 20 spores/ml; 200 spores/ml; 2000 spores/ml;20,000 spores/ml and 200,000 spores/ml in water.

1 ml spore suspension was spread on glazed and unglazed faces of 5×5 cmceramic tiles and the tiles were allowed to dry overnight at roomtemperature.

Tiles, each with 20; 200; 2000; 20,000 and 200,000 spores/tile wereplaced both the glazed side up or with the unglazed side up in 9 cmpetri dishes.

The following reagents were mixed:

-   -   222 microliter Myceliophthora thermophila laccase solution    -   702 microliter Potassium iodide solution    -   222 microliter methylsyringate solution    -   2769 microliter 1,2-propanediol    -   26586 microliter DMG buffer, pH 6.5 —and 1400 microliter of this        mixture was pipetted onto the surface of each tile and gently        spread to cover the tile from corner to corner with the pipette        tip. As a control, the spore inoculated tiles were treated with        1400 microliter of the control substance: 3 ml 1,2-propanediol        mixed with 29 ml DMG buffer pH 6.5.

The tiles were allowed to incubate, uncovered, at room temperature(approx. 24 degrees Celsius) over night. The surface of each dry tilewas covered by a thin layer of molten (approx. 45 degrees Celsius) TBBgrowth medium with agarose. When the agarose growth medium hadsolidified, the tiles were incubated in a moist chamber at 30 degreesCelsius for approx. 20 hours. Following incubation, microcolonies wererevealed by adding 3 mM MTT, drop by drop, until the agarose surface ofthe tile was covered. After ½-2 hours live micro-colonies were seen aspurple spots. In table 14 the results from a comparison of the treatedtiles with control tiles are shown. TABLE 14 Decontamination of ceramictiles seeded with Bacillus thuringiensis spores withlaccase-iodide-enhancer solution. Glazed face Unglazed face No. ofspores No. of spores germinated No. of spores No. of spores germinateddeposited Control Treated deposited Control Treated 20 approx. 20 0 20approx. 20 0 200 approx. 200 0 200 approx. 200 0 2000 too many to 0 2000too many to 0 count count 20.000 too many to 0 20.000 too many to 0count count 200.000 too many to 5 200.000 too many to 14 count count

The results demonstrate that spores deposited on surfaces areinactivated by the laccase system. The density of the surface depositedspores was approx. 4×10⁷/m².

Example 6

Killing of Spores Deposited on Ceramic Tiles II

The following reagents were prepared:

-   -   DMG buffer (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.5 with NaOH;    -   Bacillus thuringiensis spores were re-suspended in DMG buffer to        a density of 2×10⁹ spores per ml;    -   Myceliophthora thermophila laccase (as disclosed in WO 95/33836,        SEQ ID NO:1; and available from Novozymes A/S) was diluted to 30        mg/ml.    -   Polyporus pinsitus laccase (as disclosed in WO 96/00290, FIG. 1,        SEQ ID NO:1; and available from Novozymes A/S) was diluted to        7.5 mg/ml.    -   200 mM Potassium iodide (KI) solution in water.    -   10 mM Methylsyringate (methyl 3,5-dimethoxy-4-hydroxybenzoate,        Sigma S40,944-8) solution in ethanol/DMG buffer (1:1);    -   3 mM MTT (3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium        bromide, Sigma M2128) solution in water.

TBB growth medium with agarose:

-   -   10 g/l Tryptose,    -   3 g/l Beef Extract,    -   5 g/l NaCl,    -   5 g/l Agarose    -   water ad 1000 ml    -   final pH 7.2+/−0.2.

Spores were diluted to approximately 5×10⁶ spores/ml in water.

1 ml spore suspension was spread on each glazed and unglazed face of 5×5cm ceramic tiles, and the tiles were allowed to dry overnight at roomtemperature.

The tiles were arranged with the spore impregnated side up in 9 cm petridishes.

Mixtures A, B, C and D were prepared by adding together the followingreagents:

A

-   -   5 microliter Myceliopthora thermophila laccase solution    -   70 microliter Potassium iodide solution    -   25 microliter methylsyringate solution    -   270 microliter 1,2-propanediol    -   2600 microliter DMG buffer, pH 6.5    -   yielding a mixture with 50 microgramme/ml Myceliopthora        thermophila laccase.

B

-   -   2.5 microliter Myceliopthora thermophila laccase solution    -   70 microliter Potassium iodide solution    -   25 microliter methylsyringate solution ‘270 microliter        1,2-propanediol    -   2600 microliter DMG buffer, pH 6.5    -   yielding a mixture with 25 microgramme/ml Myceliopthora        thermophila laccase.

C

-   -   20 microliter Polyporus pinsitus laccase solution    -   70 microliter Potassium iodide solution    -   25 microliter methylsyringate solution    -   270 microliter 1,2-propanediol    -   2600 microliter DMG buffer, pH 6.5    -   yielding a mixture with 50 microgramme/ml Polyporus pinsitus        laccase.

D

-   -   10 microliter Polyporus pinsitus laccase solution    -   70 microliter Potassium iodide solution    -   25 microliter methylsyringate solution    -   270 microliter 1,2-propanediol    -   2600 microliter DMG buffer, pH 6.5    -   yielding a mixture with 25 microgramme/ml Polyporus pinsitus        laccase.

1400 microliter of the above-mentioned mixtures A, B, C or D werepipetted onto the surfaces of the tiles and gently spread to cover thetile from corner to corner with the pipette tip.

As a control, the spore inoculated tiles were treated with 1400microliter of the control substance: 270 microliter 1,2-propanediolmixed with 2700 microliter DMG buffer pH 6.5.

The tiles were allowed to incubate, uncovered, at room temperature(approx. 21 degrees Celsius) over night. The surface of each dry tilewas covered by a thin layer of molten (approx. 45 degrees Celsius) TBBgrowth medium with agarose. When the agarose growth medium hadsolidified, the tiles were incubated in a moist chamber at 30 degreesCelsius for approx. 20 hours. Following incubation, microcolonies wererevealed by adding 3 mM MTT, drop by drop, until the agarose surface ofthe tile was covered. After ½-2 hours live micro-colonies were seen aspurple spots. In table 15 the results from a comparison of the treatedtiles with control tiles are shown. TABLE 15 Decontamination of ceramictiles seeded with Bacillus thuringiensis spores withlaccase-iodide-enhancer solution with decreasing amounts of laccaseMixture A Mixture B Mixture C Mixture D Control Face of (Number of(Number of (Number of (Number of (Number of ceramic tile colonies)colonies) colonies) colonies) colonies) Glazed face 10 11 0 1 too manyto count Unglazed 24 approx. 100 2 4 too many to face count

The results demonstrate that spores deposited on surfaces areinactivated by laccase decontamination systems with low (less than 50mg/l) laccase concentrations.

Example 7

Killing of Spores Deposited on Textile

The following reagents were prepared:

-   -   Spores were re-suspended in sterile water to a density of        6×10⁸/ml.    -   DMG buffer (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.5 with NaOH;    -   DMG buffer (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.0 with NaOH;    -   Myceliophthora thermophila laccase (as disclosed in WO 95/33836,        SEQ ID NO:1; and available from Novozymes A/S) was diluted to 30        milligram per ml in DMG buffer;    -   200 mM Potassium iodide (KI) solution in water;    -   10 mM Methylsyringate (methyl 3,5-dimethoxy-4-hydroxybenzoate,        Sigma S40,944-8) solution in Ethanol/DMG buffer (1:1);    -   0.1% (v/v) Tween    -   3 mM MTT (3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium        bromide, Sigma M2128) solution in water.

TBB growth medium:

-   -   10 g/l Tryptose,    -   3 g/l Beef Extract,    -   5 g/l NaCl,    -   water ad 1000 ml    -   final pH 7.2+/−0.2.

Laccase-iodide-enhancer solution:

-   -   37 microliter Myceliopthora thermophila laccase solution    -   115 microliter Potassium iodide solution    -   37 microliter methylsyringate solution    -   500 microliter 1,2-propanediol    -   4311 microliter DMG buffer, pH 6.0

100 microliter of the spore suspension was pipetted onto three dryPro-shot Gun cleaning cotton patches, 1⅛″×1⅛″. The patches (patch 1,patch 2 and control) were allowed to dry overnight at room temperature.

Each of patch 1 and patch 2 were placed in a 5 cm open Petri dish and 2ml of the laccase-iodide-enhancer solution was poured onto the patch andthe open Petri dish with the patch was allowed to incubate at roomtemperature (approx. 24 degrees Celsius) for 24 hours. A patch (control)treated with a 500 microliter 1,2-propanediol in 4500 microliter DMGbuffer pH 6.5 was used as a control.

The almost-dry patches were transferred to 50 ml screwcapped disposablecentrifuge tubes containing 10 ml 0.1% (v/v) Tween. The tubes wereimmersed in an ultrasound (Branson) cleaning bath for 30 minutes at roomtemperature.

100 microliter of the fluid from the ultrasound treated centrifuge tubeswere pipetted to wells in row A in a microtiter plate according to table16. TABLE 16 Microtiter plate setup. Laccase-iodide- Laccase-iodide-enhancer enhancer Patch 1 Patch 2 Control Wells (microliter)(microliter) (microliter) A1-A4 100 0 0 A5-A8 0 100 0 A9-12 0 0 100

180 microliter TBB growth medium was added to all wells in rows B to Hof the microtiter plate. Serial 10 fold dilutions were made by pipetting20 microliter from row A to row B, and then from row B to row C, andthen from row C to row D, and so on until row H. Then 150 microliter TBBwas pipetted into the wells in row A

The microtiter plate was incubated at 30 degrees Celsius for 12-24 hoursto allow spores to germinate and grow. Growth was evaluated by amicroplate reader and visually by “developing the growth” by addition of5 microliter 3 mM MTT to each well. Formation of purple formazansreveals bacterial growth and thus the degree of spore inactivation.TABLE 17 Results of evaluation of growth. 1 2 3 4 5 6 7 8 9 10 11 12 A −− − − − − − − + + + + B + + + + − − − − + + + + C − − − − − − −− + + + + D − − − − − − − − + + + + E − − − − − − − − + + − − F − − − −− − − − − − + + G − − − − − − − − − − − − H − − − − − − − − − − − −

The results in Table 17 show that only the spore containing patchestreated with both laccase, potassium iodide and enhancing agent(methylsyringate) are effectively disinfected.

Example 8

Thiosulphate Quenching of the Sporocidal Effect

The following reagents were prepared:

-   -   DMG buffer (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.5 with NaOH;    -   Bacillus thuringiensis Spores were re-suspended in DMG buffer to        a density of 2×10⁹ spores per ml;    -   Myceliophthora thermophila laccase (as disclosed in WO 95/33836,        SEQ ID NO:1; and available from Novozymes A/S) was diluted to        6000 microgram per ml in DMG buffer;    -   200 mM Potassium iodide (KI) solution in water;    -   1 mM Methylsyringate (methyl 3,5-dimethoxy-4-hydroxybenzoate,        Sigma S40,944-8) solution in ethanol/DMG buffer (1:1);    -   Sterile water;    -   10% (WN) sodium thiosulphate;    -   3 mM MTT (3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium        bromide, Sigma M2128) solution in water.

TBB growth medium:

-   -   10 g/l Tryptose,    -   3 g/l Beef Extract,    -   5 g/l NaCl,    -   water ad 1000 ml    -   final pH 7.2+/−0.2.

Spore suspension was pipetted into the wells in row A of 5 microtiterplates. The other reagents were added as indicated in table 18 below.The reaction was initiated by the addition of laccase solution. Themicrotiter plates was then preincubated at 30 degrees Celsius for thespecified times: one for 15 minutes, one for 30 minutes, one for 1 hour,one for 2 hours and one for 22 hours.

At the end of the preincubation 50 microliter 10% (w/v) sodiumthiosulphate was added to each well in row A and the plate was allowedto incubate a further 60 minutes at room temperature (approx. 24 degreesCelsius). TABLE 18 Microtiter plate setup. Sodium- DMG Methyl-Thiosulphate Water buffer Spores Laccase KI syringate (microliter)(microliter) Wells (microliter) (microliter) (microliter) (microliter)(microliter) (*) (*) A1-3 123 50 8 6.25 12.5 50 0 A4-6 123 50 8 6.2512.5 0 50 A7-9 150 50 0 0 0 50 0 A10-12 150 50 0 0 0 0 50(*) Added after 15 and 30 minutes and after 1, 2 and 22 hourspreincubation.

180 microliter TBB growth medium was added to all wells in rows B to Hof the microtiter plate. Then serial 10 fold dilutions were made bypipetting 20 microliter from row A to row B, and then from row B to rowC, and then from row C to row D, and so on until row H.

The microtiter plates were incubated at 30 degrees Celsius for 20-24hours to allow spores to germinate and grow. Growth was evaluated by amicroplate reader and visually by “developing the growth” by addition of5 microliter 3 mM MTT to each well. Formation of purple formazansreveals bacterial growth and thus the degree of spore inactivation. Thedifference in growth between the control without laccase and the laccasekilling mixture, where growth can be detected, directly gives thekilling potential in log units. TABLE 19 Results of evaluation ofgrowth. 15 minutes preincubation followed by quenching. 1 2 3 4 5 6 7 89 10 11 12 A B + + + + + + + + + + + + C + + + + + + + + + + + + D + + +− − + + + + + + + E + + + − − − + + + + + + F + − + − − − + + + + + + G− − − − − − + − + − + + H − − − − − − − − − − + −

TABLE 20 Results of evaluation of growth. 1 hours preincubation. 1 2 3 45 6 7 8 9 10 11 12 A B + + + + − − + + + + + + C + + + + − − + + + + + +D − + − − − − + + + + + + E − − − − − − + + + + + + F − − − − −− + + + + + + G − − − − − − + + + + + + H − − − − − − − − + − − −

TABLE 21 Results of evaluation of growth. 2 hours preincubation 1 2 3 45 6 7 8 9 10 11 12 A B − + + + + − + + + + + + C − − − − − − + + + + + +D − − − − − − + + + + + + E − − − − − − + + + + + + F − − − − −− + + + + + + G − − − − − − + + − + − + H − − − − − − − − − − − −

TABLE 22 Results of evaluation of growth. 4 hours preincubation. 1 2 3 45 6 7 8 9 10 11 12 A B − − − − − − + + + + + + C − − − − − − + + + + + +D − − − − − − + + + + + + E − − − − − − + + + + + + F − − − − −− + + + + + + G − − − − − − − + + + + + H − − − − − − − − − − − −

TABLE 23 Results of evaluation of growth. 22 hours preincubation. 1 2 34 5 6 7 8 9 10 11 12 A B − − − − − − + + + + + + C − − − − −− + + + + + + D − − − − − − + + + + + + E − − − − − − + + + + + + F − −− − − − + + + + + + G − − − − − − − + − + + + H − − − − − − − − − − + −

Thiosulphate is known to oxidise iodide very efficiently. The sporeinactivation patterns demonstrates that incubation of spores with thelaccase system for more than 1 hour, results in irreversible sporeinactivation.

Example 9

NaOH Quenching of the Sporocidal Effect

The following reagents were prepared:

-   -   DMG buffer (3,3-DiMethylGlutaric acid, Sigma D4379), 50 mM, pH        adjusted to 6.5 with NaOH;    -   Bacillus thuringiensis spores were re-suspended in DMG buffer to        a density of 2×10⁹ spores per ml;    -   Myceliopthora thermophila laccase (as disclosed in WO 95/33836,        SEQ ID NO: 1; and available from Novozymes A/S) was diluted to        6000 microgram per ml in DMG buffer;    -   200 mM Potassium iodide (KI) solution in water;    -   1 mM Methylsyringate (methyl 3,5-dimethoxy-4-hydroxybenzoate,        Sigma S40,944-8) solution in ethanol/DMG buffer (1:1);    -   Sterile water;    -   0.5 M Sodium hydroxide (NaOH);    -   0.5 M Hydrochloric Acid (HCl)    -   3 mM MTT (3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium        bromide, Sigma M2128) solution in water.

TBB growth medium:

-   -   10 g/l Tryptose,    -   3 g/l Beef Extract,    -   5 g/l NaCl,    -   water ad 1000 ml    -   final pH 7.2+/−0.2.

Spore suspension was pipetted into the wells in row A of five microtiterplates. The other reagents were added as indicated in table 24 below.The reaction was initiated by the addition of laccase solution. The fivemicrotiter plates were then preincubated at 30 degrees Celsius for 15minutes, 30 minutes, 1 hour, 2 hours and 22 hours.

At the end of the incubation 25 microliter 0.5 M sodium hydroxide wasadded to specified wells in row A, se table 23. Following a furtherincubation period of 60 minutes the added NaOH was neutralized by theaddition of 25 microliter 0.5 M HCl. TABLE 24 Microtiter plate setup.DMG Methyl- NaOH Water HCl buffer Spores Laccase KI syringate (micro-(micro- (micro- (micro- (micro- (micro- (micro- (micro- liter) liter)liter) Wells liter) liter) liter) liter) liter) (*) (*) (**) A1-3 123 508 6.25 12.5 25 0 25 A4-6 123 50 8 6.25 12.5 0 50 0 A7-9 150 50 0 0 0 250 25 A10-12 150 50 0 0 0 0 50 0(*) Added after 15, 30 minutes and after 1 hour 2 hours and 22 hourspreincubation.(**) Added after 60 minutes incubation with NaOH.

180 microliter TBB growth medium was added to all wells in rows B to Hof the microtiter plate. Then serial 10 fold dilutions were made bypipetting 20 microliter from row A to row B, and then from row B to rowC, and then from row C to row D, and so on until row H.

The microtiter plates were incubated at 30 degrees Celsius for 20-24hours to allow spores to germinate and grow. Growth was evaluated by amicroplate reader and visually by “developing the growth” by addition of5 microliter 3 mM MTT to each well. Formation of purple formazansreveals bacterial growth and thus the degree of spore inactivation TABLE25 Inactivation of spores by the laccase system. 15 minutespreincubation 1 2 3 4 5 6 7 8 9 10 11 12 A B + + + + + + + + + + + +C + + + + + + + + + + + + D + + + + + + + + + + + + E + + + − +− + + + + + + F + − − − − − + + + + + + G − − − − − − + + + + − − H − −− − − − − − − − − −

TABLE 26 Inactivation of spores by the laccase system. 1 hourpreincubation 1 2 3 4 5 6 7 8 9 10 11 12 A B + + + + + + + + + + + +C + + + + − − + + + + + + D + − + − − + + + + + + + E − − − − −− + + + + + + F − − − − − − + + + + + + G − − − − − − + + + + + + H − −− − − − − − + − + −

TABLE 27 Inactivation of spores by the laccase system. 2 hourspreincubation 1 2 3 4 5 6 7 8 9 10 11 12 A B + + + − − − + + + + + + C− + − − − − + + + + + + D − − − − − − + + + + + + E − − − − −− + + + + + + F − − − − − − + + + + + + G − − − − − − + + + + + − H − −− − − − + − − − − −

TABLE 28 Inactivation of spores by the laccase system. 4 hourspreincubation 1 2 3 4 5 6 7 8 9 10 11 12 A B − − + − − − + + + + + + C −− − − − − + + + + + + D − − − − − − + + + + + + E − − − − −− + + + + + + F − − − − − − + + + + + + G − − − − − − + + + + + − H − −− − − − + − + − − −

TABLE 29 Inactivation of spores by the laccase system. 22 hourspreincubation. 1 2 3 4 5 6 7 8 9 10 11 12 A B − − − − − − + + + + + + C− − − − − − + + + + + + D − − − − − − + + + + + + E − − − − −− + + + + + + F − − − − − − + + + + + + G − − − − − − − + − + + + H − −− − − − − − − − + −

Incubating spores (preincubated with laccase system) with analkalihydroxide reverses the inactivation to some extent. The longer thelaccase system acts on the spores the greater the nonreversibleinactivation is obtained. Preincubation for more than 4 hourspractically renders the spores unable to germinate.

1. A sporocidal composition comprising a laccase or a compoundexhibiting laccase activity, a source of oxygen, a source of iodide ionsand an enhancing agent.
 2. The composition of claim 1, wherein thesource of iodide ions is one or more salts of iodide.
 3. The compositionof claim 1, which further comprises a surfactant.
 4. An enzymatic methodof killing or inactivating spores, comprising contacting the spores witha laccase or a compound exhibiting laccase activity, a source of oxygen,a source of iodide ions and an enhancing agent.
 5. The method of claim4, wherein the source of iodide ions is one or more salts of iodide. 6.The method of claim 4, which further comprises contacting the sporeswith a surfactant.
 7. The method of claim 4, wherein the spores arelocated on a surface.
 8. The method of claim 7, wherein the surface is atextile surface.
 9. The method of claim 7, wherein the surface is asurface of laboratory or process equipment.
 10. A method ofdecontaminating a location, which has been exposed to spores, comprisingcontacting the spores with a laccase or a compound exhibiting laccaseactivity, a source of oxygen, a source of iodide ions and an enhancingagent.
 11. The method of claim 10, wherein the source of iodide ions isone or more salts of iodide.
 12. The method of claim 10, which furthercomprises contacting the spores with a surfactant.
 13. A containercomprising the composition of claim 1, wherein the components of thecomposition are packaged in one or more compartments or layers.
 14. Aready-to-use sporocidal formulation comprising the composition ofclaim
 1. 15. (canceled)